Preclinical metabolism and the disposition of vornorexant/TS-142, a novel dual orexin 1/2 receptor antagonist for the treatment of insomnia.

We investigated the metabolism and disposition of vornorexant, a novel dual orexin receptor antagonist, in rats and dogs, and clarified in vitro metabolite profiles in humans. Furthermore, we investigated the pharmacokinetics of active metabolites in rats and dogs and their CNS distribution in rats to elucidate its contribution to drug efficacy. [14 C]vornorexant was rapidly and mostly absorbed after the oral administration in rats and dogs. The drug-derived radioactivity, including metabolites, was distributed to major organs such as the liver, kidneys in rats, and was almost eliminated within 24 h post-dose in both species. Metabolite profiling revealed that main clearance mechanism of vornorexant was metabolism via multiple pathways by oxidation. The major circulating components were the cleaved metabolites (M10, M12) in rats, and the unchanged form in dogs, followed by M1, and then M3. Incubation with human hepatocytes resulted in formation of metabolites, including M1, M3, M10, and M12. The metabolic pathways were similar in all tested species. Resulting from the PK and CNS distribution of active metabolites (M1 and M3) with weaker pharmacological activity, the concentration of the unchanged form was higher than that of active metabolites in rat CSF and dog plasma, suggesting that the unchanged form mainly contributed to the drug efficacy. These findings demonstrate that vornorexant is absorbed immediately after administration, and vornorexant and its metabolites are rapidly and completely eliminated in rats and dogs. Thus, vornorexant may have favorable pharmacokinetic profiles as a hypnotic drug to provide rapid onset of action and minimal next-day residual effects in humans.

Preclinical Metabolism and Disposition of TP0473292, a Novel Oral Prodrug of the Potent Metabotropic Glutamate 2/3 Receptor Antagonist TP0178894 for the Treatment of Depression.

TP0473292 is an adamantane carboxylic acid (ACA) ester prodrug for enhancing the oral bioavailability of the hydrophilic glutamate analog TP0178894, a novel metabotropic glutamate 2 and 3 receptor antagonist, and being developed as an antidepressant. TP0473292 showed high membrane permeability and rapid hydrolysis to TP0178894 in rat, monkey, and human liver S9 fractions, with a conversion rate of such that complete conversion by first-pass metabolism was expected. TP0473292 was also hydrolyzed in the intestinal, renal, and lung S9 fractions, coinciding with the result that TP0473292 was activated by carboxylesterase (CES) 1 and more efficiently by CES2. Despite the rapid hydrolysis of TP0473292 in the intestinal S9 fraction, TP0473292 achieved good oral bioavailability of poorly permeable TP0178894 (approximately 60%) in rats and monkeys, with no TP0473292 detected in the plasma, revealing that rapid hydrolysis in the intestine is not necessarily a disadvantage. We also confirmed the penetration of TP0178894 into the cerebrospinal fluid and its unmetabolized excretion in urine. The ester promoiety, ACA, was metabolized to chemically stable acyl glucuronide and excreted in urine in rats and monkeys, suggesting a low risk of idiosyncratic drug toxicity. TP0473292 and its metabolites did not show a drug-drug interaction potential via cytochrome P450 in humans. These results suggested that TP0473292 functions as an ideal oral prodrug in humans; this was later confirmed to be true in phase 1 clinical trials. Furthermore, ACA was firstly confirmed to be a useful promoiety for hydrophilic drugs to enhance their oral bioavailability. SIGNIFICANCE STATEMENT: Hydrolysis in the intestine reportedly has negative effects on the oral bioavailability of hydrophilic active metabolites of ester prodrugs. This study reports the preclinical pharmacokinetics of a hydrophilic metabotropic glutamate 2/3 receptor antagonist, TP0178894, and its ester prodrug TP0473292, which was found to act as an oral prodrug despite being activated predominantly in the intestine. Furthermore, this study firstly reports that adamantane carboxylic acid is useful as the ester promoiety of a prodrug for increasing lipophilicity and oral bioavailability.

Chiral LC-MS/MS method for the simultaneous determination of (R,S)-ketamine, (R,S)-norketamine, and (2R,6R;2S,6S)-hydroxynorketamine in mouse plasma and brain.

A convenient LC-MS/MS assay method to simultaneously and sensitively determine (R,S)-ketamine (Ket), (R,S)-norketamine (NK), and (2R,6R;2S,6S)-hydroxynorketamine (HNK) enantiomers in plasma and brain from mice was developed. This method enables the chiral separations of these six enantiomers in one analysis by constructing a column-switching system composed of one achiral column and two chiral columns with a relatively short analysis time (17 min). The chromatography involves the separation of (2R,6R;2S,6S)-HNK from (R,S)-Ket and (R,S)-NK on an octadecyl-silica column, followed by chiral separations on a CHIRALPAK AY-RH column for (2R,6R;2S,6S)-HNK or on a CHIRALPAK AS-RH column for the other analytes. The calibration curves for plasma and brain showed a good linearity in the range of 3-1000 ng/mL and 1.5-500 ng/g, respectively. The accuracy ranged from 90.0% to 104.0% in within-run and between-run. This validated method was applicable to determine the stereoselective pharmacokinetic profiles of (R,S)-Ket, (R,S)-NK, and (2R,6R;2S,6S)-HNK in plasma and brain collected from individual mice after a single intraperitoneal dosing of racemic Ket at an antidepressant dose. It is hoped that this assay will greatly help for understanding the relationship between the antidepressant actions of (R,S)-Ket enantiomers or their metabolites and their pharmacokinetics.

Elucidation of clearance mechanism of TP0463518, a novel hypoxia-inducible factor prolyl hydroxylase inhibitor: does a species difference in excretion routes exist between humans and animals?

1. TP0463518, a novel hypoxia-inducible factor prolyl hydroxylase inhibitor, is reportedly excreted predominantly through urinary excretion in an unchanged form in humans, with partial biliary excretion also possible. However, the clearance mechanisms remain unclear. The aim of this study was to investigate the clearance mechanisms in humans and to assess species differences in the excretion routes.2. TP0463518 was not metabolised in rat, dog, or human hepatocytes. TP0463518 is a substrate for human BCRP, OATP1B1, OATP1B3, and OAT3, suggesting that renal uptake by OAT3 is probably the predominant clearance route, with hepatic uptake by OATP1B1 and OATP1B3 contributing partially to clearance in humans.3. A species difference in excretion routes was observed. The unchanged urinary excretion rates in humans, male rats, female rats, dogs, and monkeys were 80.7%, 0.1%, 40.9%, 15.2%, and 72.6%, respectively. Urinary excretion was predominant in humans and monkeys, while only biliary excretion was observed in male rats. Uptake studies using hepatocytes showed that the hepatic uptake clearance in rats was 13.6-fold higher than that in humans. Therefore, not only reabsorption via renal tubules, but also hepatic uptake seems to be involved in the species differences in excretion routes between rats and humans.

Bottom-up physiologically based pharmacokinetic modelling for predicting the human pharmacokinetic profiles of the ester prodrug MGS0274 and its active metabolite MGS0008, a metabotropic glutamate 2/3 receptor agonist.

For ester prodrugs that are used to improve the gastrointestinal absorption of highly hydrophilic, pharmacologically active substances, it is challenging to predict the human pharmacokinetics (PK) of the prodrugs and their parent compounds using only preclinical data.This research was aimed at constructing a PBPK model for predicting the human PK of the ester prodrug MGS0274 and its parent compound MGS0008 after a single oral administration of MGS0274 besylate.First, we identified carboxylesterase 1 (CES1) as the major enzyme involved in the hydrolysis of MGS0274. Second, we constructed a new compartment model to estimate the passive diffusion clearance (CLpd) of MGS0008, a critical parameter for predicting the PK of highly hydrophilic compounds, based on in vivo monkey PK data. Finally, we constructed a permeability-limited liver PBPK model incorporating the CLpd assumed to be the same in humans.We confirmed that our method reliably predicted the human PK and that the estimated CLpd was comparable to that calculated retrospectively using the PBPK model, suggesting that the methodology for estimating the CLpd was valid.Our proposed methodology is expected to be helpful for human PK prediction of ester prodrugs hydrolysed by CES1 and their hydrophilic parent compounds even during the preclinical phase.

Guaiazulene derivative 1,2,3,4-tetrahydroazuleno[1,2-b] tropone reduces the production of ATP by inhibiting electron transfer complex II.

Molecularly targeted therapy has been used for treatment of various types of cancer. However, cancer cells often acquire resistance to molecularly targeted drugs that inhibit specific molecular abnormalities, such as constitutive activation of kinases. Even in cancer cells that have acquired resistance, enhanced anabolism, including the synthesis of nucleotides, amino acids and lipids, is common to normal cancer cells. Therefore, there is a renewed interest in effectively eliminating cancer cells by specifically targeting their abnormal energy metabolism. Multiple strategies are currently being developed for mitochondrial-targeted cancer therapy, with agents targeting oxidative phosphorylation, glycolysis, the tricarboxylic acid cycle and apoptosis. In this study, we found that one of the guaiazulene derivatives, namely, 1,2,3,4-tetrahydroazuleno[1,2-b] tropone (TAT), inhibited the proliferation of cancer cell lines stronger than that of normal cells. In addition, we showed that TAT inhibited energy production in cancer cell lines, resulting in apoptosis. Analyses done in cancer cell lines and in the animal model Caenorhabditis elegans suggested that TAT acts on the mitochondrial electron transfer complex II and suppresses cellular energy production by inhibiting oxidative phosphorylation across species. These results suggest that TAT could represent a novel anticancer agent that selectively targets mitochondria.

Downregulation of CYP1A2, CYP2B6, and CYP3A4 in Human Hepatocytes by Prolyl Hydroxylase Domain 2 Inhibitors via Hypoxia-Inducible Factor-α Stabilization.

Hypoxia-inducible factor (HIF) is associated with the expression of CYP, but the underlying mechanism remains uncertain. In this study, we investigated the effect of HIF-α stabilization caused by novel prolyl hydroxylase domain (PHD) 2 inhibitors, which are HIF-α stabilizers that mimic hypoxia, on the expressions of CYP1A2, CYP2B6, and CYP3A4 in human hepatocytes. An mRNA expression analysis of human hepatocytes treated with PHD2 inhibitors for 72 hours showed the downregulation of genes encoding CYP1A2, CYP2B6, and CYP3A4. The mRNA repressions were accompanied with an increase in erythropoietin protein, a marker of HIF-α stabilization, indicating that HIF-α stabilization was involved in the downregulation of the CYP isoforms. To understand the underlying mechanisms, we assessed the relationship between the expressions of the CYP isoforms and those of their regulating transcription factors [aryl hydrocarbon receptor (AhR), AhR nuclear translocator (ARNT), constitutive androstane receptor (CAR), pregnane X receptor (PXR), and retinoid X receptor (RXR)] in human hepatocytes treated with the HIF-α stabilizers. As a result, the mRNA level of AhR did not decrease, although ARNT expression was repressed. On the other hand, the mRNA expression levels of CAR, PXR, and RXR were repressed and closely associated with those of CYP2B6 and CYP3A4. Although the underlying mechanism of the downregulation for CYP1A2 remains unclear, the presently reported results suggest that the downregulation of CYP2B6 and CYP3A4 via HIF-α stabilization is caused by a decrease in the expressions of CAR, PXR, and RXR. SIGNIFICANCE STATEMENT: We showed that hypoxia-inducible factor (HIF)-α stabilization downregulates CYP1A2, CYP2B6, and CYP3A4 using prolyl hydroxylase domain 2 inhibitors, which are HIF-α stabilizers, as a new tool to mimic hypoxia in human hepatocytes. To understand the underlying mechanisms, we assessed the relationship between the expressions of the CYP isoforms and those of their regulating transcription factors. Our findings would contribute to a better understanding of the hypoxia-triggered regulatory mechanism of drug-metabolizing enzymes in human hepatocytes.

Discovery of MGS0274, an ester prodrug of a metabotropic glutamate receptor 2/3 agonist with improved oral bioavailability.

We previously reported that MGS0008 is a selective group II metabotropic glutamate receptor (mGlu2/3 receptor) agonist that is effective in animal models of schizophrenia. MGS0008 is a highly hydrophilic glutamate analog and is therefore expected to show low oral bioavailability in humans. To improve the oral bioavailability of MGS0008, ester prodrugs of MGS0008 were synthesized and their usefulness was evaluated. Among the prodrugs, the l-menthol-ester prodrug 4h demonstrated preferable lipophilicity, good chemical stability, and a high conversion rate to MGS0008 in human and monkey liver microsomes. A pharmacokinetic study in monkeys revealed that the oral bioavailability of MGS0008 after oral dosing of compound 4h was approximately 15-fold higher than that after oral dosing of MGS0008. Based on these findings, a diastereomer of compound 4h (compound 4j, or MGS0274), was selected as a candidate for clinical drug development, and its besylate is currently under development for the treatment of schizophrenia (Development code: TS-134).

Preclinical disposition of MGS0274 besylate, a prodrug of a potent group II metabotropic glutamate receptor agonist MGS0008 for the treatment of schizophrenia.

MGS0274 besylate is an ester-based lipophilic prodrug of a metabotropic glutamate (mGlu) 2 and mGlu3 receptor agonist MGS0008 and being developed for the treatment of schizophrenia. We investigated the disposition of these compounds in rats and monkeys and in vitro metabolism in humans to evaluate whether MGS0274 besylate could be useful as a prodrug in humans. After the oral administration of MGS0274 besylate to monkeys (2.89 mg/kg), MGS0008 was immediately found in plasma, reached a maximum concentration at 4 hours postdose, and decreased with a terminal half-life of 16.7 hours; MGS0274 was barely detectable. The oral bioavailability as MGS0008 was 83.7%, which was approximately 20-fold greater than that after oral dosing of MGS0008 (3.8%). In rats, MGS0008 penetrated the cerebrospinal fluid and was eliminated slower than from plasma. The in vitro metabolism study indicated that MGS0274 was rapidly hydrolyzed to MGS0008, which was not further metabolized. After the intravenous administration of MGS0008 to rats and monkeys, almost all the dose was excreted unchanged in urine. These results suggested that MGS0274 was, as expected, presystemically hydrolyzed to MGS0008 after gastrointestinal absorption and that MGS0008 was distributed throughout the body without further metabolism and ultimately excreted in urine in the animals. Furthermore, the hydrolytic activity against MGS0274 in the human liver S9 fraction was comparable to that in monkeys, suggesting the possibility of the rapid presystemic hydrolysis of MGS0274 to MGS0008 in humans, as it is in monkeys. Consequently, MGS0274 besylate is expected to function as a preferable prodrug in humans.

Systematic synthetic study of four diastereomerically distinct limonene-1,2-diols and their corresponding cyclic carbonates.

In order to produce versatile and potentially functional terpene-based compounds, a (R)-limonene-derived diol and its corresponding five-membered cyclic carbonate were prepared. The diol (cyclic carbonate) comprises four diastereomers based on the stereochemical configuration of the diol (and cyclic carbonate) moiety. By choosing the appropriate starting compounds (trans- and cis-limonene oxide) and conditions, the desired diastereomers were synthesised in moderate to high yields with, in most cases, high stereoselectivity. Comparison of the NMR data of the obtained diols and carbonates revealed that the four different diastereomers of each compound could be distinguished by reference to their characteristic signals.

Is the History Repeated? Can (2R,6R)-Hydroxynorketamine be Another Antidepressant?

Historically, identification of active metabolites has contributed to drug discovery for psychiatric disorders. It has led to the identification of new medications such as desipramine (a metabolite of imipramine) and paliperidone (a metabolite of risperidone). (R,S)-Ketamine, which has been regarded as the greatest breakthrough in depression research, is rapidly and stereoselectively metabolized into a variety of metabolites. Therefore, identification of an active substance after administration of (R,S)-ketamine is a critical issue, not only to delineate the underlying mechanisms but also to pave the way to develop a new antidepressant. Recently, one of the metabolites of (R,S)-ketamine, namely, (2R,6R)-hydroxynorketamine (HNK) was proposed as an active metabolite formed after administration of (R,S)-ketamine, and even as being essential for (R,S)-ketamine to exert its antidepressant effects. However, this is still controversial. Indeed, we demonstrated that the antidepressant effect of (2R,6R)-HNK is not as potent as that of its parent compounds ((R)-ketamine and (R,S)-ketamine), and that (2R,6R)-HNK is not essential for (R)-ketamine to exert its antidepressant effects. From the historical point of view, however, there is potential to discover new medications by further investigations of (2R,6R)-HNK. Therefore, more careful and thorough investigation of (2R,6R)-HNK is needed for the discovery of more efficacious and safer antidepressants.

Lack of deuterium isotope effects in the antidepressant effects of (R)-ketamine in a chronic social defeat stress model.

RATIONALE: (R,S)-ketamine, an N-methyl-D-aspartate receptor (NMDAR) antagonist, exhibits rapid and long-lasting antidepressant effects and anti-suicidal ideation in treatment-resistant patients with depression. However, the precise mechanisms underlying the antidepressant actions of (R,S)-ketamine are unknown. Although the previous report demonstrated the deuterium isotope effects in the antidepressant actions of (R,S)-ketamine, the deuterium isotope effects in the antidepressant actions of (R)-ketamine, which is more potent than (S)-ketamine, are unknown. METHODS: We examined whether deuterium substitution at the C6 position could affect antidepressant effects of (R)-ketamine in a chronic social defeat stress (CSDS) model. RESULTS: Pharmacokinetic studies showed that levels of (2R,6R)-d1-hydroxynorketamine [(2R,6R)-d1-HNK], a final metabolite of (R)-d2-ketamine, in the plasma and brain after administration of (R)-d2-ketamine (10 mg/kg) were lower than those of (2R,6R)-HNK from (R)-ketamine (10 mg/kg), indicating deuterium isotope effects in the production of (2R,6R)-HNK. In contrast, levels of (R)-ketamine and its metabolite (R)-norketamine in the plasma and brain were the same for both compounds. In a CSDS model, both (R)-ketamine (10 mg/kg) and (R)-d2-ketamine (10 mg/kg) showed rapid and long-lasting (7 days) antidepressant effects, indicating no deuterium isotope effect in the antidepressant effects of (R)-ketamine. CONCLUSIONS: The present study suggests that deuterium substitution of hydrogen at the C6 position slows the metabolism from (R)-ketamine to (2R,6R)-HNK in mice. In contrast, we did not find the deuterium isotope effects in terms of the rapid and long-lasting antidepressant effects of (R)-ketamine in a CSDS model. Therefore, it is unlikely that (2R,6R)-HNK is essential for antidepressant effects of (R)-ketamine.

Imaging Mass Microscopy of Kidneys from Azithromycin-Treated Rats with Phospholipidosis.

Drug-induced phospholipidosis is a lysosomal storage disorder characterized by the excess accumulation of tissue phospholipids. Although azithromycin can be used to induce phospholipidosis, no experimental studies evaluating the relationship between drug accumulation and phospholipid localization have been performed. In this study, azithromycin was orally administered to rats for 7 days, and the relationship between drug and phospholipid accumulation was performed using imaging mass microscopy. The administration of azithromycin induced tubular epithelial vacuolation in the inner stripe of the outer medulla of the kidney, consistent with the lamellar bodies that are typical manifestations of drug-induced phospholipidosis. Azithromycin and phospholipid tissue levels were extensively elevated in the kidneys of azithromycin-treated rats. Imaging mass microscopy revealed that both azithromycin and its metabolites were found in the kidneys of azithromycin-treated rats but not in control animals. The vacuolated areas of the kidneys were primarily found in the inner stripe of the outer medulla, consistent with the areas of high azithromycin concentration. Azithromycin was colocalized with several phospholipids-phosphatidylinositol (18:0/20:4), phosphatidylethanolamine (18:0/20:4 and 16:0/20:4), and possibly didocosahexaenoyl (C22:6)-bis(monoacylglycerol) phosphate, a putative biomarker of drug-induced phospholipidosis. In summary, we found correlations between regions of kidney damage and the accumulation of azithromycin, its metabolites, and phospholipids using imaging mass microscopy. Such analyses may help reveal the mechanism and identify putative biomarkers of drug-induced phospholipidosis.

AMPA Receptor Activation-Independent Antidepressant Actions of Ketamine Metabolite (S)-Norketamine.

BACKGROUND: Ketamine, an N-methyl-D-aspartate receptor antagonist, exerts robust antidepressant effects in patients with treatment-resistant depression. The precise mechanisms underlying ketamine's antidepressant actions remain unclear, although previous research suggests that alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) activation plays a role. We investigated whether (S)-norketamine and (R)-norketamine, the two main metabolites of (R,S)-ketamine, also play a significant role in ketamine's antidepressant effects and whether the effects are mediated by AMPAR. METHODS: Cellular mechanisms of antidepressant action of norketamine enantiomers were examined in mice. RESULTS: (S)-Norketamine had more potent antidepressant effects than (R)-norketamine in inflammation and chronic social defeat stress models. Furthermore, (S)-norketamine induced more beneficial effects on decreased dendritic spine density and synaptogenesis in the prefrontal cortex and hippocampus compared with (R)-norketamine. Unexpectedly, AMPAR antagonists did not block the antidepressant effects of (S)-norketamine. The electrophysiological data showed that, although (S)-norketamine inhibited N-methyl-D-aspartate receptor-mediated synaptic currents, (S)-norketamine did not enhance AMPAR-mediated neurotransmission in hippocampal neurons. Furthermore, (S)-norketamine improved reductions in brain-derived neurotrophic factor-tropomyosin receptor kinase B signaling in the prefrontal cortex of mice susceptible to chronic social defeat stress, whereas the tropomyosin receptor kinase B antagonist and a mechanistic target of rapamycin inhibitor blocked the antidepressant effects of (S)-norketamine. In contrast to (S)-ketamine, (S)-norketamine did not cause behavioral abnormalities, such as prepulse inhibition deficits, reward effects, loss of parvalbumin immunoreactivity in the medial prefrontal cortex, or baseline gamma-band oscillation increase. CONCLUSIONS: Our data identified a novel AMPAR activation-independent mechanism underlying the antidepressant effects of (S)-norketamine. (S)-Norketamine and its prodrugs could be novel antidepressants without the detrimental side effects of (S)-ketamine.

No Sex-Specific Differences in the Acute Antidepressant Actions of (R)-Ketamine in an Inflammation Model.

Background: Although previous reports suggest sex-specific differences in the antidepressant actions of (R,S)-ketamine, these differences in the antidepressant actions of (R)-ketamine, which is more potent than (S)-ketamine, are unknown. Methods: Saline or (R)-ketamine was administered 23 hours post lipopolysaccharide administration to adult male or female mice. Subsequently, antidepressant effects were assessed using a forced swimming test. Furthermore, the concentration of (R)-ketamine and its 2 major metabolites, (R)-norketamine and (2R,6R)-hydroxynorketamine, was measured in the plasma and brain after the administration of (R)-ketamine in the mice. Results: (R)-ketamine (10 mg/kg) significantly attenuated the increased immobility time of forced swimming test in the lipopolysaccharide-treated mice. There were no sex-specific differences in the concentrations of (R)-ketamine and its 2 metabolites in the plasma and brain. Conclusions: These findings showed no sex-specific differences in terms of the acute antidepressant effects and pharmacokinetic profile of (R)-ketamine.

(2R,6R)-Hydroxynorketamine is not essential for the antidepressant actions of (R)-ketamine in mice.

(R,S)-Ketamine has rapid and sustained antidepressant effects in depressed patients. Although the metabolism of (R,S)-ketamine to (2 R,6 R)-hydroxynorketamine (HNK), a metabolite of (R)-ketamine, has been reported to be essential for its antidepressant effects, recent evidence suggests otherwise. The present study investigated the role of the metabolism of (R)-ketamine to (2 R,6 R)-HNK in the antidepressant actions of (R)-ketamine. Antidepressant effects were evaluated using the forced swimming test in the lipopolysaccharide (LPS)-induced inflammation model of mice and the tail suspension test in naive mice. To prevent the metabolism of (R)-ketamine to (2 R,6 R)-HNK, mice were pretreated with cytochrome P450 (CYP) inhibitors. The concentrations of (R)-ketamine, (R)-norketamine, and (2 R,6 R)-HNK in plasma, brain, and cerebrospinal fluid (CSF) samples were determined using enantioselective liquid chromatography-tandem mass spectrometry. The concentrations of (R)-norketamine and (2 R,6 R)-HNK in plasma, brain, and CSF samples after administration of (R)-norketamine (10 mg/kg) and (2 R,6 R)-HNK (10 mg/kg), respectively, were higher than those generated after administration of (R)-ketamine (10 mg/kg). Nonetheless, while (R)-ketamine attenuated, neither (R)-norketamine nor (2 R,6 R)-HNK significantly altered immobility times of LPS-treated mice. Treatment with CYP inhibitors prior to administration of (R)-ketamine increased the plasma levels of (R)-ketamine, while generation of (2 R,6 R)-HNK was almost completely blocked. (R)-Ketamine exerted the antidepressant effects at a lower dose in the presence of CYP inhibitors than in their absence, which is consistent with exposure levels of (R)-ketamine but not (2 R,6 R)-HNK. These results indicate that metabolism to (2 R,6 R)-HNK is not necessary for the antidepressant effects of (R)-ketamine and that unmetabolized (R)-ketamine itself may be responsible for its antidepressant actions.

A rapid and sensitive chiral LC-MS/MS method for the determination of ketamine and norketamine in mouse plasma, brain and cerebrospinal fluid applicable to the stereoselective pharmacokinetic study of ketamine.

A novel method for the rapid and sensitive chiral determination of ketamine and norketamine in mouse plasma, brain and cerebrospinal fluid (CSF) was developed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). This method reduces the required matrix volume, compared with a previously reported chiral assay method for ketamine and norketamine. The method involves the deproteinization of a small amount of biological matrix (corresponding to 5µL of plasma, 10mg of brain, or 2.5µL of CSF) using a water-miscible organic solvent containing 2H4-norketamine as an internal standard, the direct injection of the organic supernatant into an LC-MS/MS system, chiral separation on a CHIRALPAK AS-3R column (4.6mm i.d.×100mm, 3µm particles), and detection by electrospray ionization-selected reaction monitoring with an analytical run time of 5min. The lower limits of quantification for ketamine and norketamine enantiomers were 1ng/mL (plasma), 0.5ng/g (brain) and 2ng/mL (CSF). A good linearity of the calibration curves was obtained within a range of 1000-fold. The newly developed method was successfully used to determine the concentrations of ketamine and norketamine in mouse samples (plasma, brain and CSF) in a stereoselective manner. Therefore, this method is expected to contribute to the elucidation of the roles of ketamine and its metabolites in the antidepressant actions of ketamine.

Antidepressant Potential of (R)-Ketamine in Rodent Models: Comparison with (S)-Ketamine.

The rapid-acting and long-lasting antidepressant effects of (R,S)-ketamine have recently gained much attention. Although (S)-ketamine has been studied as an active isomer, recent evidence suggests that (R)-ketamine exhibits longer-lasting antidepressant effects than (S)-ketamine in rodents. However, the antidepressant potential of (R)-ketamine has not been fully addressed. In the present study, we compared the antidepressant effects of (R)-ketamine with those of (S)-ketamine in animal models of depression, including a model that is refractory to current medications. Both (R)-ketamine and (S)-ketamine exhibited antidepressant effects at 30 minutes as well as at 24 hours after administration in forced-swimming and tail-suspension tests in mice. At 48 hours after administration, however, (R)-ketamine still exerted a significant antidepressant effect in the tail-suspension test, whereas the effect of (S)-ketamine was no longer observed. Moreover, (R)-ketamine, but not (S)-ketamine, significantly reversed the depressive-like behavior induced by repeated treatments with corticosterone in rats at 24 hours after a single administration. This effect was attenuated by an α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor antagonist, suggesting the involvement of AMPA receptor stimulation in the effects. Both (R)-ketamine and (S)-ketamine exhibited practically the same exposure levels in plasma, brain, and cerebrospinal fluid in mice and rats, and both compounds were rapidly eliminated from plasma (<4-8 hours). The present results confirmed the previous findings that (R)-ketamine exerted longer-lasting antidepressant effects than (S)-ketamine in animal models of depression. Moreover, our study is the first to demonstrate that (R)-ketamine exerted a sustained antidepressant effect even in a model that is refractory to currently prescribed antidepressants.

In vitro evaluation of potential drug interactions mediated by cytochrome P450 and transporters for luseogliflozin, an SGLT2 inhibitor.

1. We evaluated potential in vitro drug interactions of luseogliflozin, a sodium-glucose cotransporter 2 (SGLT2) inhibitor, mediated by CYP inhibition, CYP induction and drug transporters using human liver microsomes, primary hepatocytes and recombinant cells-expressing efflux or uptake transporters, respectively. 2. Human CYP inhibition studies indicated that luseogliflozin was a weak inhibitor for CYP2C19 with an IC50 value of 58.3 µM, whereas it was not an inhibitor of the other eight major isoforms that were tested. The exposure of primary hepatocytes to luseogliflozin for 72 hrs weakly induced CYP3A4 at a concentration of 10 µM, whereas it did not induce CYP1A2 or CYP2B6 at concentrations of 0.1-10 µM. 3. An in vitro transport study suggested that luseogliflozin is a substrate for human P-glycoprotein (P-gp), but not for breast cancer resistance protein (BCRP), organic anion transporting polypeptide (OATP) 1B1 and OATP1B3, organic anion transporter (OAT) 1 and OAT3, or organic cation transporter (OCT) 2. Luseogliflozin weakly inhibited OATP1B3 with an IC50 value of 93.1 µM, but those for other transporters are greater than 100 µM. 4. Based on the therapeutic plasma concentration of the drug, clinically relevant drug interactions are unlikely to occur between luseogliflozin and coadministered drugs mediated by CYPs and/or transporters.